Name: quantitative multispectral analysis following fluorescent tissue transplant for visualization of cell origins, types, and interactions
Uploaded: 2013-09-22T20:30:00
Description: Watch this Scientific Journal Video about Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions at JoVE.com

Vi er taknemmelige for diskussioner, vejledning og støtte fra Drs. Michael Andreeff MD, PhD., Og Jared Burks PhD. fra MD Anderson Flowcytometri og Cellular Imaging Core Facility. Dette arbejde blev støttet delvist af tilskud fra National Cancer Institute (RC1-CA146381, CA-083.639, R01NS06994, CA116199 og CA109451 for FCM. ELS understøttes også af hæren Department of Defense (BC083397).

Syngene murine knoglemarvstransplantation (BMT), som er godkendt af institutionelle IACUC protokoller (Bemærk: Succes for denne protokol kræver udnyttelse af enhver mærket celletype (r) som mål for multispektrale analyse, og for at lette den efterfølgende analyse, kan man udnytte enhver genetisk eller stabil cellulær mærkning. Vi foreslår, at enhver celle, væv eller organ-til-være kan anvendes til en transplantation model og at transplantationen kan indeholde så mange unikke mærkede populati...

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Heri beskriver vi anvendelsen af ​​Multispektralbilledteknik vi betegner som MIMicc-multispektral interrogering af Multiplexed cellulære sammensætninger til at analysere knoglemarv afledte stromale komponenter i tumormikromiljøet, men denne metode og koncept kan anvendes til at dechifrere andre cellulære elementer, der udgør en kompleks væv, såsom dem, der ses under sårhelingskomplikationer reaktioner eller under en regenerativ væv. I disse forsøg udnytter vi transgene mus fluorescens skelne knoglemarv-afl...

Forståelse væv udvikling og reparation er væsentligt at belyse deltagende cellulære komponenter i sårheling, 2,3 regenerativ medicin, udviklingsmæssige biologi og tumor biologi. Under omstændighederne i reparation, mange celletyper infiltrere det omgivende mikromiljø til støtte i vaskularisering, ECM deposition, spredning og omstrukturering væv. Cellulære faktorer og fænotyper kan identificeres på grundlag af multiparameter, multiplex markører, der kan identificere lokaliseringen, differentiering status og samspil mellem cellulære komponenter i undersøgte mikromiljø. Heri beskriver vi tumor udvikling som et prototypisk eksempel på denne flerfarvet-flercellede transplantation model efterfulgt af Multispektralbilledteknik og spektral unmixing metodologi. Tumorprogression er en flertrins proces, der er præget af flere erhvervede funktioner, der omfatter forbedret spredning, enntiapoptotic invasive og angiogene egenskaber. 4. Tumor udvikling fremmes af ikke-neoplastiske celler, der rekrutteres til det omgivende miljø for at tilvejebringe vækstfaktorer, strukturelle matricer, vaskulære netværk og immunmodulation. 1,5,6 Denne microhabitat består af celler, der stammer fra lokale, tilstødende væv, såsom fedt og blodkar og fjerne kilder såsom knoglemarv afledte celler 1. Omfanget af ikke-neoplastisk celle inkorporering afhænger af efterspørgslen fra tumor, der ofte svarer til trin / karakteren af ​​tumor. For at forstå den rolle, tumor støttende mikromiljø, må man forstå oprindelsen og differentiering potentiale af de ikke-neoplastiske cellepopulationer. Denne protokol er blevet designet til at støtte i fortolkningen af ​​tumor progression gennem visualisering af både knoglemarv afledte cellulære komponenter, og den lokale væv derived celler. Utilizing fluorescerende reportergenassays-udtrykkende transgene mus vi transplanterede GFP (grønt fluorescerende protein) knoglemarv i en letalt bestrålet RFP (rødt fluorescerende protein) mus. Efter en vellykket knoglemarv transplantation, er en syngen tumorcellelinje injiceres orthotopisk og lov til at indpode i 4-8 uger. Den resulterende tumor udskæres fra musen og forarbejdet til immunfluorescens (IF) farvning at visualisere stromale komponenter. Multiplexing IF markører er en almindeligt anvendt teknik, der involverer væsentlig optimering 7-9, men ved hjælp af en Multispektralbilledteknik / unmixing platform forbedrer mulighederne for fluorescerende markør kombinationer, der besidder spektral overlapning. Heri præsenterer vi en teknik, vi kalder MIMicc-multispektral forhør af Multipleksede cellulære kompositioner at plette og analysere op til otte markører i en tumor sektion på et enkelt dias for at analysere de cellulære oprindelse, cellulær differentiering strater og celle-celle interaktioner af komponenter i tumor mikromiljø. Denne forenklede eksempel har potentialet til at blive udbygget med henblik på at analysere fem, seks eller flere markører udnytte antistoffer eller intracellulære promotordrevet fluorescerende udtryk. Tabel 1 viser potentielle fluorescerende antistoffarvning kombinationer med passende arter tages i betragtning.

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td></td>
		 </tr>
		 <tr>
			<td>.2 &mu;m filter</td>
			<td >Fisher</td>
			<td>09-740-35A</td>
			<td></td>
		 </tr>
		 <tr>
			<td>10 ml lure lock </td>
			<td >BD</td>
			<td>309604</td>
			<td></td>
		 </tr>
		 <tr>
			<td>25 G needle</td>
			<td >Cardinal</td>
			<td>BF305122</td>
			<td></td>
		 </tr>
		 <tr>
			<td>40nm filter</td>
			<td >BD</td>
			<td>352340</td>
			<td></td>
		 </tr>
		 <tr>
			<td>50 ml conical tube</td>
			<td >Fisher</td>
			<td>1495949A</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Alexafluor 488-ms1</td>
			<td >invitrogen</td>
			<td>A21121</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Alexafluor 594-Rb</td>
			<td >invitrogen</td>
			<td>A21207</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Alexafluor 647-ch</td>
			<td >Invitrogen</td>
			<td>A21449</td>
			<td></td>
		 </tr>
		 <tr>
			<td>BSA</td>
			<td >Sigma</td>
			<td>A7906-500G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Cover slips</td>
			<td >Corning</td>
			<td>2940-243</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CRI-Nuance</td>
			<td>Caliper Life Sciences</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dapi</td>
			<td >Invitrogen</td>
			<td>D1306</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td >CellGro</td>
			<td>10-017-CV</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ethanol</td>
			<td >Dharmacon</td>
			<td>4004-DV</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FBS</td>
			<td >invitrogen</td>
			<td>16000-044</td>
			<td></td>
		 </tr>
		 <tr>
			<td>GFP-ms1</td>
			<td >Abcam</td>
			<td>ab38689</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Insulin needle</td>
			<td >BD</td>
			<td>329424</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Maleic acid</td>
			<td >Acros</td>
			<td>100-16-7</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Moisture chamber box</td>
			<td >Evergreen</td>
			<td>240-9020-Z10</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mounting media</td>
			<td >dako</td>
			<td>S3023</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Nail hardener</td>
			<td >Sally Hansen</td>
			<td>2103</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pap pen</td>
			<td >Abcam</td>
			<td>Ab2601</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pen/Strep L Glut</td>
			<td >invitrogen</td>
			<td>10378016</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Steril PBS</td>
			<td >invitrogen</td>
			<td>14040-182</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin</td>
			<td >invitrogen</td>
			<td>252000-56</td>
			<td> </td>
		 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>
 Blocking Buffer	
	<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td>maleic acid</td>
 <td>14.51 g </td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>10.95 g</td>
 </tr>
 <tr>
 <td>NaOH pellets (to 7.5pH)</td>
 <td>9+g</td>
 </tr>
 <tr>
 <td>Distilled water</td>
 <td>250 ml</td>
 </tr>
 </tbody>
 </table>
	*add 0.2% Tween-20 to 1x solution for blocking buffer with 2%BSA/2%FBS. Stir and filter. 
	Antigen retrieval buffers:
	<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td colspan="2">Tris-EDTA Buffer (10 mM Tris Base, 1 mM EDTA Solution, 0.05% Tween 20, pH 9.0)</td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>1.21 g</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>0.37 g</td>
 </tr>
 <tr>
 <td>Distilled water</td>
 <td>1000 ml (100 ml to make 10x, 50 ml to make 20x)</td>
 </tr>
 <tr>
 <td>Tween 20</td>
 <td>0.5 ml</td>
 </tr>
 </tbody>
 </table>
	*Store at RT for 3 months or at 4 &deg; C for longer
	<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td colspan="2">Sodium Citrate Buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0)</td>
 </tr>
 <tr>
 <td>Tri-sodium citrate (dihydrate)</td>
 <td>2.94 g</td>
 </tr>
 <tr>
 <td>Distilled water</td>
 <td>1000 ml</td>
 </tr>
 <tr>
 <td>HCl (adjust pH to 6.0)</td>
 <td>1N</td>
 </tr>
 <tr>
 <td>Tween 20</td>
 <td>0.5 ml</td>
 </tr>
 </tbody>
 </table>
 </td>
 </tr>
		</tbody>
 </table>

quantitative multispectral analysis following fluorescent tissue transplant for visualization of cell origins, types, and interactions

Med ønsket om at forstå bidragene af multiple cellulære elementer for udviklingen af ​​et komplekst væv, såsom de talrige celletyper, der deltager i regenererende væv tumordannelse eller vaskulogenese vi udtænkt en flerfarvet cellulær transplantation model af tumorudvikling i som cellepopulationer stammer fra forskellige fluorescens farvede reportergenassays mus og er transplanteret, indpodet eller injiceres i og omkring et udviklingsland tumor. Disse farvede celler bliver derefter rekrutteret og indarbejdet i tumorstroma. For at kvantitativ vurdering knoglemarv afledte stromale tumorceller, vi transplanterede GFP udtrykke transgene hel knoglemarv ind i dødeligt bestrålede RFP udtrykker mus som er godkendt af IACUC. 0ovarian tumorer, der blev orthotopisk injiceret i transplanterede mus blev udskåret 6-8 uger efter transplantation og analyseret for knoglemarv markør for oprindelse (GFP) samt antistofmarkørerne at detektere tumorassocieretstroma hjælp multispektrale billeddannelse teknikker. Vi derefter tilpasset en metode vi kalder MIMicc-Multispektral Interrogation af Multipleksede cellulære kompositioner, ved hjælp af multispektrale unmixing af fluoroprobes til kvantitativt vurdere, hvilke mærket celle kom fra, hvilke start befolkninger (baseret på originale reportergenassays etiketter), og som vores evne til at unmix 4, 5 , 6 eller flere spektre per dias stiger, har vi tilføjet ekstra immunhistokemi forbundet med celleafstamninger eller differentiering for at øge præcision. Udnytte software til at registrere co-lokaliserede multiplexede-fluorescerende signaler, tumor stromale befolkninger kan spores, opregnede og karakteriseret baseret på markør farvning. 1.

Ved hjælp af en Multispektralbilledteknik teknik til at analysere tumorer transplanteret i vores transgene BMT musemodel, vi er i stand til at skelne stromal tumor komponenter, der er knoglemarv oprindelse. Den oprindelige BMT blev bekræftet tre uger efter transplantationen ved flowcytometri (figur 1). Orthotopisk injicerede umærkede æggestokkræft efter BMT engraftment bekræftelser blev udskåret og fikseret i formalin 6 uger efter første tumor injektion. Paraffinindlejret tumorsnit blev snittet ...

Watch this Scientific Journal Video about Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions at JoVE.com

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

Komplekse væv masserne, fra organer til tumorer, er sammensat af forskellige cellulære elementer. Vi belyst bidrag cellefænotyper indenfor et væv udnytte multi-mærkede fluorescerende transgene mus i kombination med multiparameter immunfluorescensfarvning efterfulgt af spektral unmixing at dechifrere celle oprindelse samt celle egenskaber baseret på protein udtryk.

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